Method of detecting lung disease

ABSTRACT

A method of faciltating the obtaining of a mucus sample from at least one lung of a subject comprises administering a physiologically acceptable salt to at least one lung of the subject in an amount effective to hydrate lung mucus secretions therein, and concurrently administering to said at least one lung of the subject, in an amount effective to hydrate lung mucous secretions therein, uridine triphosphate, an active analog thereof, or a pharmaceutically acceptable salt of either thereof. Pharmaceutical compositions useful for carrying out the method comprise, in combination, a physiologically acceptable salt, and uridine triphosphate, an active analog thereof, or a pharmaceutically acceptable salt of either thereof. The composition may be a liquid composition or a dry powder composition.

This invention was made with Government support under grant numberHL-SPO1-34322 from the National Institutes of Health (NIH). TheGovernment has certain rights to this invention.

FIELD OF THE INVENTION

This application concerns lung diagnostic assays in general, andparticularly concerns a lung diagnostic assay in which lung mucussecretions are hydrated to facilitate collection thereof.

BACKGROUND OF THE INVENTION

The analysis of sputum samples is particularly important in thetreatment and diagnosis of many lung disorders, including lung cancerand tuberculosis (TB).

In particular, microbial infections of the lung are a serious problem inpatients afflicted with acquired immune deficiency syndrome (AIDS). Twoparticularly problematic infections are Pneumocystis carinii pneumoniainfections and mycobacterial infections.

Pneumocystis carinii pneumonia infections are typically referred to as"PCP" infections. It is now estimated that approximately 70 percent ofpatients afflicted with AIDS will contract this disease. PCP may betreated with pentamidine isethionate, but an unfortunate side effect ofthis treatment is its toxicity. Accordingly, there is a continued needfor techniques which permit the rapid and convenient screening of AIDSpatients for this disease, and for the rapid, early, and accuratediagnosis thereof.

Mycobacteria are a large, diverse, and widely distributed family ofaerobic, nonsporulating, nonmotile bacilli that have a high cell-walllipid content and a slow growth rate. Some Mycobacteria are harmless,while others are significant pathogens. The pathogenic Mycobacteriuminclude M. tuberculosis, responsible for tuberculosis, as well asnon-tuberculosis Mycobacteria such as M. avium, responsible forMycobacterium Avium complex infections.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of faciltating theobtaining of a mucus sample from at least one lung of a subject. Themethod comprises administering a physiologically acceptable salt to atleast one lung of the subject in an amount effective to hydrate lungmucus secretions therein, and concurrently administering to said atleast one lung of the subject, in an amount effective to hydrate lungmucous secretions therein, a compound of Formula (I) below, or apharmaceutically acceptable salt thereof: ##STR1## wherein:

X₁, X₂, and X₃ are each independently selected from the group consistingof OH and SH;

R₁ is selected from the group consisting of O, imido, methylene, anddihalomethylene; and

R₂ is selected from the group consisting of H and Br.

The method is accompanied or followed by the step of collecting a mucussample from said at least one lung of said subject (e.g., by saidsubject expectorating a sputum sample).

A second aspect of the present invention is pharmaceutical compositionuseful for facilitating the collecting of a mucus sample from at leastone lung of a subject, said composition comprising, in combination, aphysiologically acceptable salt in an amount effective to hydrate lungmucus secretions; and a compound of Formula (I) as given above, or apharmaceutically acceptable salt thereof, in an amount effective tohydrate lung mucus secretions. The composition may be a liquidcomposition or a dry powder composition.

DETAILED DESCRIPTION OF THE INVENTION

Compounds illustrative of the compounds of Formula (I) above (alsoreferred to as "active compounds" herein) include: (a) uridine5'-triphosphate (UTP); (b) uridine 5'-O-(3-thiotriphosphate) (UTPγS);and (c) 5-bromo-uridine 5'-triphosphate (5-BrUTP). One preferredcompound of Formula (I) above is the UTP analog uridine5'-O-(3-thiotriphosphate) (or "UTPγS"). These compounds are known or maybe made in accordance with known procedures, or variations thereof whichwill be apparent to those skilled in the art. See generally U.S. Pat.No. 5,292,498 to Boucher; N. Cusack and S. Hourani, Annals N.Y. Acad.Sci. 603, 172-181 (G. Dubyak and J. Fedan Eds. 1990). For example, UTPmay be made in the manner described in Kenner et al., J. Chem. Soc.1954, 2288; or Hall and Khorana, J. Chem. Soc. 76, 5056 (1954). SeeMerck Index, Monograph No. 9795 (11th Ed. 1989). UTPγS may be made inthe manner described in G. Goody and F. Eckstein, J. Am. Chem. Soc. 93,6252 (1971).

For simplicity, Formula I herein illustrates uridine triphosphate activecompounds in the naturally occurring D configuration, but the presentinvention also encompasses compounds in the L configuration, andmixtures of compounds in the D and L configurations, unless specifiedotherwise. The naturally occurring D configuration is preferred.

The active compounds disclosed herein can be prepared in the form oftheir pharmaceutically acceptable salts. Pharmaceutically acceptablesalts are salts that retain the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; and salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; and (b) salts formed fromelemental anions such as chlorine, bromine, and iodine.

Amiloride (also known as3,5,-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide), benzamil(also known as3,5-diamino-6-chloro-N-(benzylaminoaminomethylene)pyrazinecarboxamide) and phenamil (also known as3,5-diamino-6-chloro-N-(phenylaminoaminomethylene) pyrazinecarboxamide)are known compounds and are disclosed in U.S. Pat. No. 3,313,813 to E.Cragoe. The terms "amiloride," "benzamil," and "phenamil," as usedherein (also refered to as "active compounds" herein), include thepharmaceutically acceptable salts thereof, such as (but not limited to)amiloridehydrochloride, benzamilhydrochloride or phenamil hydrochloride.Amiloride, benzamil or phenamil used to prepare compositions for thepresent invention may alternatively be in the form of a pharmaceuticallyacceptable free base of amiloride, benzamil or phenamil. Because thefree base of the compound is less soluble than the salt, free basecompositions are employed to provide more sustained release of benzamilor phenamil to the lungs.

Physiologically acceptable salts (also sometimes referred to as "activecompounds" herein) used to carry out the method of the present inventionare those that hydrate lung mucus secretions by facilitating thetransport of water from lung endothelial cells into the mucus.Physiologcally acceptable salts are salts that retain the desiredbiological activity of hydrating lung mucus secretions and do not impartundesired toxicological effects. Examples of such salts include, but arenot limited to, sodium chloride, potassium chloride, choline chloride,and N-methyl-D-glucamine chloride. Sodium chloride is currentlypreferred. These salts may be provided in the form of a dry respirablepowder (discussed below) or as an aqueous saline solution.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

The active compounds disclosed herein may be administered to the lung(s)of a subject by any suitable means. As used herein, the word"concurrently" means sufficiently close in time to produce a combinedeffect (that is, concurrently may be simultaneously, or it may be two ormore events occuring within a short time period before or after eachother). By "at least one lung" is meant that administration of activecompounds may be to one or both lungs of the subject, but whereadministration is to only one lung, then administration of the variousactive compounds is to the same lung.

Active compounds are preferably administered by administering an aerosolsuspension of respirable particles comprised of the active compound,which the subject inhales. The respirable particles may be liquid orsolid. The particles may optionally contain other therapeuticingredients such as amiloride, benzamil or phenamil, with the selectedcompound included in an amount effective to inhibit the reabsorption ofwater from airway mucous secretions, as described in U.S. Pat. No.4,501,729 (applicant specifically intends the disclosure of this and allother patent references cited herein be incorporated herein byreference).

The particulate pharmaceutical composition may optionally contain adispersant which serves to facilitate the formation of an aerosol. Asuitable dispersant is lactose, which may be blended with the activecompound or compounds in any suitable ratio (e.g., a 1 to 1 ratio byweight).

Particles comprised of active compound for practicing the presentinvention should include particles of respirable size: that is,particles of a size sufficiently small to pass through the mouth andlarynx upon inhalation and into the bronchi and alveoli of the lungs. Ingeneral, particles ranging from about 1 to 10 microns in size (moreparticularly, less than about 5 microns in size) are respirable.Particles of non-respirable size which are included in the aerosol tendto deposit in the throat and be swallowed, and the quantity ofnon-respirable particles in the aerosol is preferably minimized. Fornasal administration, a particle size in the range of 10-500 μm ispreferred to ensure retention in the nasal cavity.

Liquid pharmaceutical compositions of active compound for producing anaerosol may be prepared by combining the active compound with a suitablevehicle, such as sterile pyrogen free water. The hypertonic salinesolutions used to carry out the present invention are preferablysterile, pyrogen-free solutions, comprising from one to ten percent (byweight) of the physiologically acceptable salt, and more preferably fromthree to seven percent by weight of the physiologically acceptable salt.Where compounds of Formula (I) or the pharmaceutically acceptable saltsthereof are included in the hypertonic saline solution, they aretypically included in a concentration ranging from about 10⁻⁴ M to about10⁻¹ M, and more preferably in a concentration ranging from about 10⁻² Mto about 10⁻¹ M. Other therapeutic compounds such as amiloride, benzamilor phenamil may optionally be included (when such compounds areincluded, the saline solution is preferably not more than 0.3 percent byweight of the physiologically acceptable salt, and more preferably is0.12 percent by weight of the physiologically acceptable salt).

Solid particulate compositions containing respirable dry particles ofmicronized active compound may be prepared by grinding dry activecompound with a mortar and pestle, and then passing the micronizedcomposition through a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprised of the activecompound may optionally contain a dispersant which serves to facilitatethe formation of an aerosol. A suitable dispersant is lactose, which maybe blended with the active compound in any suitable ratio (e.g., a 1 to1 ratio by weight). Again, other therapeutic compounds such asamiloride, benzamil or phenamil may also be included.

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No.4,501,729. Nebulizers are commercially available devices which transformsolutions or suspensions of the active ingredient into a therapeuticaerosol mist either by means of acceleration of compressed gas,typically air or oxygen, through a narrow venturi orifice or by means ofultrasonic agitation. Suitable formulations for use in nebulizersconsist of the active ingredient in a liquid carrier, the activeingredient comprising up to 40% w/w of the formulation, but preferablyless than 20% w/w. The carrier is typically water (and most preferablysterile, pyrogen-free water) or a dilute aqueous alcoholic solution,preferably made isotonic with body fluids by the addition of, forexample, sodium chloride. Optional additives include preservatives ifthe formulation is not made sterile, for example, methylhydroxybenzoate, antioxidants, flavoring agents, volatile oils,buffering agents and surfactants.

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles which are respirable, as explained above, andgenerate a volume of aerosol containing a predetermined metered dose ofa medicament at a rate suitable for human administration. Oneillustrative type of solid particulate aerosol generator is aninsufflator. Suitable formulations for administration by insufflationinclude finely comminuted powders which may be delivered by means of aninsufflator or taken into the nasal cavity in the manner of a snuff. Inthe insufflator, the powder (e.g., a metered dose thereof effective tocarry out the treatments described herein) is contained in capsules orcartridges, typically made of gelatin or plastic, which are eitherpierced or opened in situ and the powder delivered by air drawn throughthe device upon inhalation or by means of a manually-operated pump. Thepowder employed in the insufflator consists either solely of the activeingredient or of a powder blend comprising the active ingredient, asuitable powder diluent, such as lactose, and an optional surfactant.The active ingredient typically comprises from 0.1 to 100 w/w of theformulation. A second type of illustrative aerosol generator comprises ametered dose inhaler. Metered dose inhalers are pressurized aerosoldispensers, typically containing a suspension or solution formulation ofthe active ingredient in a liquified propellant. During use thesedevices discharge the formulation through a valve adapted to deliver ametered volume, typically from 10 to 150 μl, to produce a fine particlespray containing the active ingredient. Suitable propellants includecertain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation mayadditionally contain one or more co-solvents, for example, ethanol,surfactants, such as oleic acid or sorbitan trioleate, antioxidants andsuitable flavoring agents.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 10 to 150liters per minute, more preferably from about 30 to 150 liters perminute, and most preferably about 60 liters per minute. Aerosolscontaining greater amounts of medicament may be administered morerapidly. Typically, each aerosol is delivered to the patient for aperiod from about five to about 20 minutes, with a delivery period ofabout ten minutes being preferred.

The dosage of the compound of Formula (I), or the pharmaceuticallyacceptable salt thereof, will vary depending on the condition beingtreated and the state of the subject, but generally may be an amountsufficient to achieve dissolved concentrations of active compound on theairway surfaces of the subject of from about 10₋₉ to about 10⁻²Moles/liter, more preferably from about 10⁻⁷ to about 10⁻³ Moles/liter,and most preferably from about 10⁻⁶ to about 10⁻⁴ Moles/liter. Dependingupon the solubility of the particular formulation of active compoundadministered, the daily dose may be divided among one or several unitdose administrations.

When the sputum sample is subjected to cytological, bacterial or DNAanalysis for detecting infectious microbial species therein, the sputumsample may first be digested with a liquefying agent, such asN-acetyl-L-cystein (NALC) and sodium hydroxide.

As noted above, the present invention is particularly useful forcollecting mucus samples which are used for detecting Pneumocystiscarinii pneumonia and Mycobacteria infections. The term "Mycobacteria"as used herein has its conventional meaning in the art referring toacid-fast, non-motile, rod shaped bacteria. See generally B. Davis etal., Microbiology, 724-742 (#d. Ed. 1980). All generally belong to thefamily Mycobacteriaceae. By way of example, the mycobacteria include,but are not limited to, Mycobacterium africanurn, M. avium, M. bovis, M.bovis-BCG, M. chelonae, M. fortuitum, M. gordonae, M. intracellulae, M.kansasii, M. leprae, M. microti, M. paratuberculosis, M. scrofulaceum,and M. tuberculosis. The present invention is useful in diagnosing bothtuberculosis and non-tuberculosis Mycobacteria, and is useful indiagnosing M. avium complex infections.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLE 1 Delivery of UTP followed by Hypertonic Saline

A subject is caused to inhale an aerosol of UTP solution (10⁻² M UTP in0.9% (by weight) sterile pyrogen-free saline solution) delivered by anOmron ultrasonic nebulizer for 10 minutes. Immediately afterwards, thesubject is caused to inhale an aerosol of hypertonic saline solution (3%sterile pyrogen-free saline solution), delivered by a DeVilbisultrasonic nebulizer for 10 minutes. During inhalation of the aerosolsand after inhalation of the aerosols, the subject is encouraged tocough, and all sputum is collected during aerosol inhalation and over a20 minute interval following cessation of aerosol inhalation. The sputumis captured in a plastic sputum container. The sputum so obtained isanalyzed for content pending the clinical requirement, includinganalyses of cytologies for pulmonary neoplasm, and the presence ofinfectious agents such Pneumocystis carinii via silver staining andimmunofluorescence techniques. The technique is applicable for thediagnoses of other micro-organisms in the lower lung, includingbacterial, viral, amd microplasma, using culture, immunocytochemical,and molecular (PCR, in situ hybridization) techniques.

EXAMPLE 2 Delivery of UTP with Amiloride

This example is carried out in essentially the same manner as example 1above, except that the UTP is dissolved in a 0.12% sterile pyrogen-freeNaCl solution, and the solution also contains 10⁻² M amiloride.

EXAMPLE 3 Delivery of Hypertonic Saline followed by UTP

This Example is carried out in essentially the same manner as Example 1above, except that the hypertonic saline solution is delivered first,and the UTP solution is delivered immediately thereafter. Durations ofdelivery and concentrations remain the same.

EXAMPLE 4 Concurrent Delivery of UTP in Hypertonic Saline

This Example is carried out by modification of Example 1 above, with10⁻¹ UTP being diluted in the hypertonic saline solutiion so that asingle solution is delivered to the patient by inhalation of an aerosolgenerated by the Omron ultrasonic nebulizer for a period of 15 minutes.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. Among other things,volumes, times, and amounts can be varied from those specifically setforth above. Accordingly, the invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of facilitating the obtaining of amucus sample from at least one lung of a subject,comprising:administering by inhalation a physiologically acceptable saltto at least one lung of said subject in an amount effective to hydratelung mucus secretions therein, wherein said physiologically acceptablesalt facilitates the removal of said lung mucus from said lung, andconcurrently administering by inhalation to said at least one lung ofsaid subject, in an amount effective to stimulate the secretion of lungmucus therein, a compound of Formula (I) below, or a pharmaceuticallyacceptable salt thereof: ##STR2## wherein: X₁, X₂, and X₃ are eachindependently selected from the group consisting of OH and SH; R₁ isselected from the group consisting of O, imido, methylene, anddihalomethylene; and R₂ is selected from the group consisting of H andBr.
 2. A method according to claim 1, wherein said physiologicallyacceptable salt is administered by administering an aqueous solutioncontaining both said salt and said compound of Formula (I) or saidpharmaceutically acceptable salt thereof.
 3. A method according to claim1, wherein said compound of Formula (I), or said pharmaceuticallyacceptable salt thereof, is administered in an amount sufficient toachieve concentrations thereof on the airway surfaces of said subject offrom about 10⁻⁹ to about 10⁻³ Moles/liter.
 4. A method according toclaim 1, further comprising concurrently administering a compoundselected from the group consisting of amiloride, benzamil and phenamil,and pharmaceutically acceptable salts thereof, to said subject in anamount effective to inhibit the reabsorption of water from lung mucoussecretions.
 5. A method according to claim 1, wherein X₂ and X₃ are OH.6. A method according to claim 1, wherein R₁ is oxygen.
 7. A methodaccording to claim 1, wherein R₂ is H.
 8. A method according to claim 1,wherein said compound is selected from the group consisting of uridine5'-triphosphate, uridine 5'-O-(3-thiotriphosphate), and thepharmaceutically acceptable salts thereof.
 9. A method according toclaim 1, wherein said physiologically acceptable salt is selected fromthe group consisting of sodium chloride, potassium chloride, cholinechloride, and N-methyl-D-glucamine chloride.
 10. A method according toclaim 1, wherein said physiologically acceptable salt is sodiumchloride.
 11. A method according to claim 1, wherein saidphysiologically acceptable salt and said compound of Formula (I) or saidpharmaceutically acceptable salt thereof are delivered by administeringan aerosol suspension of respirable particles comprised thereof to thesaid at least one lung of said subject.
 12. A method according to claim11, wherein said particles are selected from the group consisting ofsolid particles and liquid particles.
 13. A method according to claim11, wherein said aerosol is comprised of particles having a particlesize within the range of about 1 to 10 microns.
 14. A method accordingto claim 1, wherein said physiologically acceptable salt is sodiumchloride.